Smooth muscle mitogen

ABSTRACT

A novel growth factor (BTC-CG) was purified from the conditioned medium of pancreatic beta tumor cells initially derived from transgenic mice (RIPl-Tag2). The purification scheme included BioRex 70 chromatography, phenyl-Sepharose chromatography, TSL-GEL heparin FPLC and C4 reverse phase HPLC. The peptide also stimulated proliferation of bovine smooth muscle cells. It was not inactivated by boiling, by 10 mM dithiothreitol or by exposure to IM acetic acid. Biological activity of BTC-GF was recovered from a single band of protein which had a molecular weight of 2,000 on SDS-PAGE. The Partial N-terminal amino acid sequence of this protein was determined with an ABI 470A protein sequencer as: Asp-Gly-[X]-Thr-[X]-Arg-Thr-Pro-Glu-[X]-Asn-Gly.

This invention was made with U.S. Government support and the Governmenthas certain rights in the invention.

The present invention is directed to a novel growth factor whichstimulates the growth of smooth muscle cells and to the uses thereof.

BACKGROUND OF THE INVENTION

While smooth muscle cell proliferation has been extensively studied,(see, e.g. Schwartz et al., Circulation Research, Vol. 58, No. 4, page427, the disclosure of which is incorporated by reference herein), thesignals controlling the proliferation of smooth muscle cells remainlargely unknown. Smooth muscle cell replication is known to play acentral role in diseases such as arteriosclerosis (atherosclerosis andhypertension). Lack of smooth muscle proliferation in infants also playsa role in vascular malformations. This failure of smooth muscle cellreplication results in untreatable vascular lesions which often lead todeath.

Although it is now generally acknowledged that replication of smoothmuscle cells occurs during formation of atherosclerotic lesions, therole of that proliferative response in the overall history of the plaqueis not all obvious. A few investigators have suggested that replicationoccurring during development of arteries is the initial event information of atherosclerotic lesions, preceding lipid accumulation orendothelial injury.

The major hypothesis explaining smooth muscle replication in the vesselwall is the response-to-injury hypothesis. In brief, hypothesis is thatsmooth muscle cells in the wall normally exist in a quiescent state.When the endothelium is injured, platelets release a factor or factorsthat stimulate smooth muscle cell movement into and replication withinthe arterial intima (Ross Arteriosclerosis 1:293-311, 1981). Ross alsoshowed the cultured smooth muscle cells require a platelet derivedgrowth factor (PDGF) for proliferation (Ross and Glomset N. Eng. J. Med.295; 369-377 and 420-425, 1976). The apparent conclusion is thatplatelet release is necessary for smooth muscle proliferative responseto balloon denudation.

Ross's observation led to the ensuing purification of the PDGF,identification of its receptor and, more recently, identification of theoncogene c-sis as the gene for one of the two PDGF peptide chains.

The second known requirement for cell cycle progression is availabilityof somatomedin C., also known as insulin-like growth factor (IGF-1).IGF-1 itself can be synthesized by smooth muscle cells, and antibodiesto IGF-1 inhibit cell cycle progression. These data suggest that PDGF iscapable of stimulating production of its own progression factor. Thisobservation is of considerable importance to the interesting possibilitythat smooth muscle replication may be controlled by factors intrinsic tothe vessel wall.

Other substances mitogenic for smooth muscle cells, apart from PDGF havealso been studied. In addition, platelets also contain a proteinresembling epidermal growth factor (EGF) (Oka and Orth, J.Clin. Invest.72:249-259, 1983) and Assoian et al., 1984) and a factor able to assistgrowth of cell in suspension called β-tumor growth factor (Tucker etal., Science 226: 705-707, 1984). The relative contribution of each ofthese to stimulation of proliferation is largely unknown.

The stimuli controlling smooth muscle replication in hypertension alsoremains largely unknown. PDGF may play an important role inmicrovascular changes in malignant hypertension, but is not likely to beinvolved in large vessels or in any vessel affected by milder and morechronic forms of high blood pressure.

While there has been much research on the role of smooth muscle invarious disease pathologies, and several mechanisms and roles of growthfactors such as PDGF have been explored, there continues to be a needfor new information about mitogens which stimulate the proliferation ofsmooth muscle cells. The identification of such mitogens will permitvarious treatment strategies to be devised such as competitive bindingstrategies employing antibodies to the smooth muscle mitogen orcompetitive proteins which will bind to the receptors for such mitogens.Smooth muscle mitogens may also be used in the treatment of conditionssuch as vascular malformation or as a growth factor in wound/ulcerhealing.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a novelgrowth factor (hereinafter "BTC-GF") obtainable from the conditionedmedium of pancreatic tumor cells initially derived from transgenic mice(RIPl-Tag 2) in which virtually every beta cell expressed the oncogeneSV40 large T. A sample of the pancreatic tumor cells from which BTC-GFwas identified, isolated and purified has been deposited at the AmericanType Culture Collection 12301 Parklawn Drive, Rockville, MD 20852, Oct.26, 1990 under ATCC Accession No. CLL 10885.

BTC-GF of the present invention is not inactivated by boiling, by 10 mMdithiothreitol or by exposure to 1M acetic acid. The biological activityof BTC-GF is present as a single band of protein having a molecularweight of about 32,000 on SDS-PAGE. The partial N-terminal amino acidsequence of BTC-GF (SEQ 1D No:1) isAsp-Gly-Xaa-Thr-Xaa-Arg-Thr-Pro-Glu-Xaa-Asn-Gly.

A computer search based on this amino acid sequence through translatedGENBANK and NBRF Protein Database failed to reveal any similar proteins.

BTC-GF of the present invention can be used in the treatment of diseasessuch as vascular malformation as well as in the treatment ofwounds/ulcers and the like. BTC-GF may also be used to producecompetitive agents such as antibodies or false peptides. Suchcompetitive agents may be used in the treatment of diseases resultingfrom smooth cell proliferation such as atherosclerosis and hypertension.It may also be used as a diagnostic test in which an antibody to thegrowth factor can detect this factor in the blood of diabetics in whomdying or regenerating beta cells with islet are releasing the factor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the 3T3 cell growth factor activity of BTC-GF afterconcentrated serum free beta tumor cell conditioned medium is passedthrough a Biorex 70 Cation Exchange Column.

FIG. 2 illustrates the 3T3 cell growth factor activity of pooled activefractions from FIG. 1 when passed through a Phenyl-Sepharose column.

FIG. 3 illustrates the 3T3 cell growth factor activity of the pooledactive fractions from the phenyl-sepharose column when passed through anFPLC heparin affinity column.

FIG. 4 illustrates the 3T3 cell growth factor activity of the pooledactive fractions from the heparin affinity column when passed through anHPLC C4 reverse phase column.

FIG. 5 is a silver strain of BTC-GF an gel from the pooled activefractions obtained by repeating the HPLC C4 reverse phase columnpurification.

FIG. 6 illustrates the mitogenic activity of BTC-GF on bovine smoothmuscle cell.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a novelgrowth factor BTC-GF which promotes the proliferation of smooth musclecells.

BTC-GF produced in accordance with the present invention has a molecularweight of about 32,000 on SDS-PAGE and is heat stable when subjected toboiling. BTC-GF is also stable in the presence of 10 mM dithiothreitoland when exposed to IM concentration of acetic acid.

BTC-GF was identified and isolated from the conditioned medium ofpancreatic tumor cells (ATCC No. CRL 10585, a sample of which wasdeposited at the American Type Culture Collection under the BudapestTreaty on Oct. 26, 1990) were initially derived from transgenic mice(RIP1- Tag 2) in which virtually every beta cell expressed the oncogeneSV40large T.

While a number of methods may be employed in purifying BTC-GF, thepreferred method is outlined below and described in more detail in theExamples.

First, the beta tumor cells are cultured in roller bottles in DMEM with5% calf serum for four days. The medium is then replaced with serum freemedium and cultured for 48-72 hours before harvest.

Next, serum free beta tumor cell conditioned medium is concentrated andpassed through a number of columns such as a Biorex 70 column, a phenylsepharose column, and FPLC heparin affinity column, and an HPLC reversephase column.

Purified BTC-GF of the present invention has a N-terminal amino acidsequence as determined with an ABI 470A protein sequensor of SEQ ID No:1.

Purified BTC-GF in accordance with the present invention can be used inthe treatment of pathological conditions such as vascular malformationby intravascular infusion, or for the treatment of atherosclerosis byadministration of a competitive inhibitor.

Purified BTC-GF can also be used in the treatment of wounds, ulcers andthe like.

Purified BTC-GF of the present invention can also be used to producevarious competitive agents which can be used in the treatment ofatherosclerosis and hypertension. Competitive agents such as antibodiesor false proteins can be produced which will compete with and/or blockBTC-GF from stimulating proliferation of smooth muscle cells.

BTC-GF can also be used to generate antibodies to itself. The antibodygenerated can be polyclonal or monoclonal depending upon the particularapplication for which it is designed. Such antibodies can be prepared bytechniques well known to the skilled artisan. For example, the proteinor antigenic portion thereof can be conjugated to keyhole limpethemocyanin (KLH) and used to raise an antibody in an animal such as arabbit. Typically, the peptide-KLH conjugate is injected several timesover a period of about two months to generate antibodies. The antibodyis then collected from serum by standard techniques. Alternatively,monoclonal antibodies can be produced in cells which produce antibodiesto the protein by using standard fusion techniques for forming hybridomacells. [Kohler, G., et al., Nature 256:495 (1975) which is incorporatedby reference]. Typically, this involves fusing an antibody producingcell with an immortal cell line such as a myeloma cell to produce thehybrid cell. Alternatively, monoclonal antibodies can be produced fromcells by the method of Huse, et al, Science 246:1275 (1989) which isincorporated herein by reference.

The invention will be further illustrated by reference to the followingexamples which will aid in the understanding of the present invention,but which are not to be construed as a limitation thereof.

Growth factor activities discussed in the Examples and in Table 1 wereassayed by measuring the incorporation of [methyl-³ H]thymidine into DNAof quiescent mouse Balb/c 3T3 cells as previously described (Shing Y,Davidson S. and Klagsbrun M. Methods in Enzymology, 146B, 42-48, 1987)the disclosure of which is hereby incorporated by reference.

EXAMPLE 1

Primary cultures of pancreatic beta tumor cells (ATCC Accession No. CRL10585) were prepared in Dulbecco's modified Eagles medium (DMEM)containing 10% calf serum. These cultures were plated on 162 cm² cellflasks (Costar Cat #3150) and incubated in a 37° C. humidified CO₂incubator. These cells were used as a source for seeding into 900 cm²-growth-area roller bottles (Costar Cat #3901) containing 125 ml of DMEMwith 5% calf serum. The bottles were gased with 90% air/5% CO₂ androtated on a Cell Production Roller Apparatus (Bellco) at 0.5 rpm in a37° C. incubator. After 4 days the medium in each bottle was replacedwith serum-free medium. The medium was harvested and replaced with freshmedium after incubation for 48-72 hours. Six liters of conditionedmedium were collected weekly as the starting materials for thepurification of growth factors.

EXAMPLE 2 Method for the Purification of BTC-GF

Step 1. Concentration

Ten liters of serum free beta tumor cell conditioned medium wereconcentrated to 500 ml at 4° C. with an Amicon hollow fiber concentratorusing a filter of 10,000 molecular weight cutoff. The concentratedmedium was subsequently equilibrated to 50 mM NaCI, 10 mM Tris, pH 7 bycontinuous dialysis.

Step 2. BioRex 70 Chromatooraphy

The concentrated medium was applied to a BioRex column (200 ml bedvolume) equilibrated with 10 mM Tris, pH 7 at 4° C. The column wasrinsed with 400 ml of the same buffer and the biological activity wasthen eluted with a NaCl gradient from 400 ml of 0 M to 400 ml of 0.6 Mat a flow rate of 60 ml/hour (FIG. 1).

Step 3. Phenyl-Sepharose Chromatography

The active fractions from BioRex column were pooled, boiled for 5minutes and clarified by centrifugation (10,000 x g, 20 minutes). Theclear supernatant solution was brought to 1.5M (NH₄)₂ SO₄ and applied toa phenyl-Sepharose column (25 ml bed volume) equilibrated at 1.5M (NH₄)₂SO₄, 10 mM potassium phosphate buffer, pH 7 at 4° C. The column wasrinsed with 100 ml of equilibration buffer and the biological activitywas subsequently eluted with a (NH₄)₂ SO₄ gradient from 170 ml of 1.5 Mto 170 ml of 0M in 10 mM phosphate buffer at pH 7 at a flow rate of 30ml/hour (FIG. 2).

Step 4. FPLC Heoarin Affinity Chromatography

The active fractions from phenyl-Sepharose column were pooled, dialyzedand applied to TSK-GEL Heparin 5PW glass column (7.5 cm ×8 mm innerdiameter) equilibrated with 10 mM Tris, pH 7 at room temperature. Thecolumn was rinsed with 10 ml of the same buffer and the biologicalactivity was eluted with NaCl gradient from 0 to 0.3M followed byanother NaCl gradient from 0.3 to 0.6M at a flow rate of 1ml/min/fraction (FIG. 3).

Step 5. HPLC C4 Reverse Phase Chromatography

The active fractions from Heparin column were pooled and injecteddirectly into a HPLC reverse phase C4 column equilibrated with 10%acetonitrile in 0.1% TFA at room temperature. The column was rinsed with20 ml of the same solution and the biological activity was eluted with agradient of acetonitrile from 10 to 35% at a flow rate of 2 ml/min andfractions of 1.5 ml were collected (FIG. 4). This step was repeated oncein order to obtain a silver-stained single band protein on SDS PAGE(FIG. 5).

A summary of the result of purification is shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    Purification of BTC-GF                                                        Purification                                                                            Total  Total  Specific                                                                              Activity                                                                            Purification                            Step      Protein, mg                                                                          Activity, U                                                                          Activity, U/mg                                                                        Recovery                                                                            Fold                                    __________________________________________________________________________    Conditioned medium                                                                      1360   24 × 10.sup.4                                                                  1.7 × 10.sup.2                                                                  100   1                                       BioRex column                                                                           52.7   6.5 × 10.sup.4                                                                 1.2 × 10.sup.3                                                                  27    7                                       Heat, 100° C.                                                                    15.5   5 × 10.sup.4                                                                   3.2 × 10.sup.3                                                                  21    19                                      (5 min.)                                                                      Phenyl column                                                                           1.5    4 × 10.sup.4                                                                   2.8 × 10.sup.4                                                                  17    164                                     Heparin column                                                                          0.09   3 × 10.sup.4                                                                   3.3 × 10.sup.5                                                                  10    1,942                                   C4 column, 1st                                                                          0.0012*                                                                              1.4 × 10.sup.4                                                                 1.2 × 10.sup.7                                                                  5.8   70,000                                  C4 column, 2nd                                                                          0.00034*                                                                             0.98 × 10.sup.4                                                                2.9 × 10.sup.7                                                                  4     170,000                                 __________________________________________________________________________     Values were based on processing of 10 liters of conditioned medium.           Biological activity was measured by DNA Synthesis in mouse 3T3 cells.         One unit of growth factor activity is defined as the amount of growth         factor needed to stimulate halfmaximal incorporation of [methyl  .sup.3       H]thymidine into DNA.                                                         Protein mass was estimated by using A.sub.280 = 1.0 for a 1 mg/ml             solution.                                                                     *Protein mass was estimated by the intensity of silver stain compared to      that of the protein standards and amino acid analysis.                   

EXAMPLE 3 Mitogenic Acitvity of BTc-GF on Smooth Muscle Cell

The purified BTC-GF stimulated the proliferation of bovine aortic smoothmuscle cell (SMC) (FIG. 6). The mitogenic activity of BTC-GF was testedon SMC cultured in DMEM containing 1% calf serum. Four days after thetest samples were added to the cultures, the cells were trypsinized andthe numbers of cells in each well of the 24 well plates were countedwith a Coulter Counter.

The protein produced by the above-exampled purification protocol has thefollowing characteristics: BTC-GF is a polypeptide having the N-terminalamino acid sequence depicted in SEQ 10 No: 1. It has a molecular weightof 32,000 as determined by SDS polyacrylamide gel electrophoresis. Itsmitogenic activity is not inactivated by exposure to high temperature(100° C., 5 minutes), sulfhydryl reducing agent (10 mM dithiothreitol)or acidic condition (pH 2.2).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                  (vi) ORIGINAL SOURCE:                                                        (A) ORGANISM: TRANSGENIC MICE (RIP1- Tag2)                                    (G) CELL TYPE: PANCREATIC TUMOR CELL                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AspGlyXaaThrXaaArgThrProGluXaaAsnGly                                          1510                                                                      

What is claimed:
 1. A purified protein having an N-terminal amino acidsequence comprising:SEQ 1D No:1, wherein said protein stimulatedproliferation of smooth muscle cells.
 2. The purified protein of claim1, wherein the protein is obtainable from beta tumor cells ATCCAccession No. CRL
 10585. 3. The purified protein of claim 1, wherein theprotein has a molecular weight of about 32,000 on SDS-PAGE.
 4. Thepurified protein of claim 1, having at least about 50% of its activityafter heating at 100° C. for 5 minutes.
 5. The purified protein of claim1, wherein said protein is stable when exposed to 1M acetic acid.
 6. Thepurified protein of claim 1, wherein the protein is stable when exposedto 10 mM dithiothreitol.
 7. A method for producing the purified proteinof claim 1 comprising:a) concentration serum free beta tumor cellconditioned medium; b) applying the conditioned medium of step a) to aweakly acidic cation exchange column containing carboxylic acid exchangegroups on a macroreticular acrylic polymer lattice; c) applying activefractions obtained from step b) to a phenyl Sepharose column; d)applying active fractions obtained from step c) to a heparin affinitycolumn; e) applying active fractions obtained from step d) to a reversephase column; and f) collecting active fractions obtained from step e).8. The method of claim 7, wherein the purified protein produced has aspecific activity of about 2.9×10⁷ unit of growth factor activity/mg. 9.A purified polypeptide which includes the amino acid sequence of SEO IDNO:1.
 10. The purified protein of claim 1 that is in native form.